-
[show abstract]
[hide abstract]
ABSTRACT: Hepatitis C is an infectious disease affecting the liver, caused by the hepatitis C virus (HCV). HCV is an etiological agent of acute and chronic liver disease that exists throughout the world. The high genetic variability of the HCV genome is reflected by six genotypes (1 to 6). Each genotype has a characteristic geographical distribution, which is important epidemiologically. HCV is a blood-borne virus that generally circulates in low titers in the serum of infected individuals. Epidemiologic studies show that the most efficient transmission of HCV is through the transfusion of blood or blood products, the transplantation of organs from infected donors, and the sharing of contaminated needles among injection-drug users. However, fewer than half of patients with acute hepatitis C report a history of such exposure. A small number of epidemiologic studies demonstrate that perinatal, sexual, household, and occupational transmission occurs, but our understanding of the risks of transmission in these settings has been limited. The therapy for chronic hepatitis C has evolved steadily since alpha interferon was first approved for use. At present, the optimal regimen appears to be a 24- or 48-week course of a combined pegylated alpha interferon and ribavirin regimen. Currently, the combination of RNAi (LV-shIRES) with IFN-α has been proposed to prevent therapeutic resistance, and to promote enhanced antiviral activity against HCV. However, any RNAi based therapy may be years away due to off-target effects.
The Journal of Infection in Developing Countries 01/2011; 5(1):1-17. · 1.19 Impact Factor
-
Mazhar Kanak,
Mohammed Alseiari,
Prathap Balasubramanian,
Krishna Addanki,
Mayank Aggarwal, Samina Noorali,
Azima Kalsum,
Kuha Mahalingam,
Gene Pace,
Nicholas Panasik,
Omar Bagasra
[show abstract]
[hide abstract]
ABSTRACT: One of the most fascinating discoveries in biology in recent years is unquestionably the identification of the family of small, noncoding RNAs known as microRNAs (miRNAs). Each miRNA targets multiple mRNA species through recognition of complementary sequences, typically located at multiple sites within the 3 untranslated region. In animals, single-stranded miRNA binds specific messenger RNA (mRNA) by a mechanism that is yet to be fully characterized. The bound mRNA remains untranslated resulting in reduced levels of the corresponding protein; however, if the sequence match between the miRNA and its target is precise, the bound mRNA can be degraded resulting in reduced levels of the corresponding transcript. Eukaryotic genes are also regulated by triplex formation between double helix and a third small RNA or DNA molecule. Thousands of triplex-forming (TF) islands in human genomes are mapped. However, the role of TF miRNAs within the hairpin structures of miRNA and the target mRNA has not been reported. We have explored TF complexes between human miRNAs (hsa-miR) that are complementary to human immunodeficiency virus (HIV)-1 and their antiviral potential as therapeutic agents.
We downloaded mature miRNA sequences from the human miRBase Sequence Database (http://microrna.sanger.ac.uk/sequences/), and computationally analyzed miRNAs that have significant homologies to HIV-1 genome (pNL 4-3 Accession #AF324493). We developed an algorithm to look for triplex-binding motifs (C+CG and T AT) and selected 4 miRNAs with 3 negative controls. TF stability was tested by using fluorophore-labeled duplexes connected by a single hexaethylene glycol moiety, representing HIV-1 proviral motifs, and black-hole quencher-1 labeled oligonucleotides, representing miRNA.
Fifty miRNAs were discovered that showed greater than 80% homology to HIV-1, of which 4 hsa-miR that exhibited an ability to form stable triplex with double stranded-HIV-1 sequences were selected. Three negative controls were used. The TF stability of the 4 hsa-miRs and the negative controls were confirmed and measured. Stably transfected Hela-CD4+ cell lines expressing each of the hsa-miR were developed. All 4 miRNAs exhibited a significant inhibition of HIV-1 as measured by HIV-1 p24 enzyme-linked immunosorbent assay (>90%; P>0.001) when compared with the 3 negative controls. By using immunohistochemical staining with triplex binding monoclonal antibodies, significant expression of TF miRNAs was detected in the cell lines, but not in the negative controls (P<0.001).
In this study, we demonstrated for the first time that besides the well-established post-transcriptional silencing based on mRNA degradation, miRNAs may be responsible for long-term latency of HIV-1 by TF, a different mechanism. We provide a possible molecular mechanism by which HIV-1 homologous miRNAs may impart resistance to HIV-1 and suggest a new miRNA-based therapeutic strategy for HIV-1.
Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 12/2010; 18(6):532-45. · 1.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Over half of human genome contains retroelements, including retrotransposons, retroviruses, and other elements. Human endogenous retroviruses (HERVs) comprise about 8% of human genome. The products of 2 of 16 identified genes of HERV-W seem to play a pivotal role in the placentation. These 2 genes are HERV-W env glycoprotein (syncytin-1) and HERV-FRD env glycoprotein (syncytin-2). It has been shown previously that syncytin-1 mediates cell-cell fusions of cytotrophoblasts into syncytiotrophoblasts. In addition, HERV-W env contains an immunosuppressive region that may prevent rejection of a semiallogenic fetus from the mother's immune system. We analyzed 40 full-term placental tissues to localize the expression of syncytin-1-ISR by immunohistochemical staining and by reverse trancscriptase (RT) in situ polymerase chain reaction (PCR). Both the immunostaining and in situ RT-PCR showed strong expression of syncytin-1 in the syncytiotrophoblast layer from the full-term placental tissues. To further analyze the mechanism of early embryo HERV-W env activation, we utilized a HTR-8/SVneo cell line developed from first trimester human trophoblasts and subjected them to various physiologic concentrations of maternal hormones. Quantitative RT-PCR analyses demonstrated that exposure to progesterone significantly upregulated the HERV-W env expression, whereas several other hormones apparently played lesser roles. In conclusion, our findings suggest that expression of syncytin-1 (HERV-W env) in utero is expressed exclusively in the syncytiotrophoblast layer and is upregulated by progesterone.
Applied Immunohistochemistry 06/2009; 17(4):319-328. · 1.63 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Hepatitis B virus (HBV) is an etiological agent of acute and chronic liver disease existing throughout the world. The high genetic variability of HBV genome is reflected by eight genotypes (A to H), and each genotype has characteristic geographical distribution, which is important epidemiologically. Previous studies from the province of Sindh, Pakistan, have reported that genotypes A and D as prevalent HBV genotypes. The aim of the study was to investigate the prevalence of HBV genotypes in physically healthy females at two universities in Karachi, Sindh, Pakistan.
Blood was collected from a total of 4,000 healthy female volunteer students and serum samples obtained were screened for Hepatitis B surface antigen (HBsAg), and anti-HBs antibodies by immunochromatography and ELISA. Genotyping was conducted for 6 HBV genotypes (A through F). Both genotyping and sequencing data of HBV positive females are described.
Out of 4,000 volunteers, 180 (4.5%) tested positive for HBsAg and 20 (0.5%) were positive for HBs antibodies. All 180 serum samples were genotyped by PCR and sequencing analyses was conducted for 21 samples. Out of 180 HBsAg positive samples, 150 showed a single HBV D genotype infection; 29 showed co-infection of genotypes B and D; and 1 exhibited co-infection of genotypes C and D. Twenty-one representative samples were selected randomly from genotypes B, D, and C for sequencing and each isolate clustered with respective reference genotype sequence, thus validating the genotyping strategy.
Genotype D appears to be the dominant genotype prevalent in Karachi's otherwise healthy female population.
The Journal of Infection in Developing Countries 02/2008; 2(5):373-8. · 1.19 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Growth differentiation factor (GDF15) is a distant member of the transforming growth factor-beta superfamily, a diverse group of structurally related proteins that exert multiple effects on cell fate such as on cell growth and differentiation but little is known about GDF15 in these processes. Previously we observed the mature GDF15 to be associated with human prostate carcinogenesis hence prompting us to study GDF15 further. Here we report gdf15 expression both at the RNA and protein levels, in normal prostatic tissues of wild type (wt) and prostatic intraepithelial neoplasia (PIN) of transgenic (Tg) 12T-7s model mice during embryonic, postnatal, and adult prostate formation up to 15 weeks after birth. Dynamic changes in expression, at both the mRNA and protein level, correlated with cell proliferation and differentiation during distinct phases of normal mouse prostate development and alterations in the dynamics of gdf15 expression correlated with the changes in development resulting in PIN formation. Most notably mature gdf15 protein was significantly elevated during hyperplasia and PIN development. Changes in the protein levels did not always correlate well with the mRNA levels. This was more prominent during PIN than during normal prostate development suggesting that this may also be an indicator of disturbed regulation of gdf15 in PIN. We propose that gdf15 is a growth factor with dual function either promoting proliferation or growth arrest and differentiation due most likely to differences in cellular differentiation. Because of the differentiation defect in PIN its epithelium no longer responds to gdf15 by cellular growth arrest as does the normal epithelium and gdf may even stimulate proliferation. The data supports our hypothesis that GDF15 plays a role in the early stages of human prostate cancer.
Differentiation 05/2007; 75(4):325-36. · 2.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To characterize angioimmunoblastic T-cell lymphoma (AILT) on morphological, immunohistochemical and molecular grounds and its association with Epstein-Barr virus (EBV) in Pakistani patients.
Case series.
Histopathology section of the Department of Pathology and Microbiology, The Aga Khan University Hospital, Karachi from January 01, 1992 to December 31, 2002.
Over a period of 11 years archival biopsy material of 13 AILT cases (lymph nodes), identified on the basis of histological and immunohistochemical criteria, using REAL and WHO classifications, were retrieved from the files of Department of Pathology. Immunophenotyping was carried out by using CD45 (LCA), two T-cell markers CD45RO (UCHL1; monoclonal) and CD3 (polyclonal). Polymerase chain reaction (PCR) was used to assess T-cell clonality for T-cell receptor (TCR)-b, g and immunoglobulin heavy chain (IgH) for FR2 and FR3 regions using primers recognizing conserved sequences of the variable (V), diversity (D) and joining (J) region segments. Association of EBV in AILT cases was studied by PCR and in situ hybridization (ISH).
This study showed AILT to constitute 0.71% of all NHLs (non-Hodgkin's lymphoma) [both T and B]. Immunohistochemical study revealed that the tumor cells were positive for CD45 (LCA), CD45RO (UCHL1) and CD3. All the 13 cases were largely negative for CD20 (L26), a B-cell marker, except few large scattered cells labelling. DNA extracted from all 13 lymph nodes was amplified using polymerase chain reaction (PCR). PCR technique demonstrated clonal gene rearrangement of the TCR-b, g and IgH regions in 3 (23.1%), 7 (53.8%) and 3 (23.1%) AILT cases, respectively out of 13 cases. Association of EBV was seen in 11 out of 13 cases (84.6%) of AILT by PCR. By ISH the prevalence of EBV was detected in 8 (88.8%) out of 9 cases.
The prevalence of AILT in the Pakistani population is slightly lower compared to other studies and that EBV is an etiological agent in pathogenesis of this disease.
Journal of the College of Physicians and Surgeons--Pakistan: JCPSP 08/2005; 15(7):404-8. · 0.34 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Anaplastic large cell lymphoma (ALCL), CD30+, is a subtype of T-non-Hodgkin's lymphoma (NHL). Its most common form is a classical systemic type that involves multiple nodal and extranodal sites. In this study, morphologic, immunohistologic, and genetic studies were performed on ALCL cases in Pakistani patients. The median age of the patients in this study was 45 years (age range: 5-70 years), with a male to female ratio of 3.4:1. Thirty-seven (37) patients were diagnosed to have Ki-1 (CD30+) ALCL, which constituted 2% of all NHLs and 12.6% of all T-NHLs, over a period of 11 years (January 01, 1992-December 31, 2002). The tumors were of either T- or null-cell type with constant (100%) expression of CD30 (Ki-1). The majority of the cases (89.2%) expressed EMA, whereas 40.5% of the cases expressed either CD45 (LCA), CD45RO (UCHL1), or ALK. The mean age of ALCL patients with null-cell phenotype was 33.8 years as compared to those with T-cell phenotype having a mean age of 36.3 years. Out of the 37 cases diagnosed as ALCL, amplifiable DNA was isolated from 28 cases, which were further assessed for T-cell clonality for T-cell receptor (TCR)-beta, gamma, and immunoglobulin heavy chain (IgH) for the FR2 and FR3 regions. The polymerase chain reaction (PCR) technique demonstrated clonal rearrangement of the TCR beta, gamma, and IgH regions in 15 (53.6%), 11 (39.3%), and 2 (7.1%) ALCL cases, respectively, out of 28 cases. Association of Epstein-Barr virus (EBV) was noted in seven out of 28 cases (25%) of ALCL by PCR, whereas ISH for EBV-encoded nuclear RNA-1 (EBER-1) detected the presence of EBV in two (16.7%) out of 12 cases, where one was T-cell ALCL and the other null-cell ALCL. Immunostaining for LMP-1 could not be performed, because tissue material was not available. In conclusion, our study demonstrated that the prevalence of ALCL in Pakistan is comparable to that reported for some of the Asian communities and by the International Lymphoma Study Group and that EBV could be partly responsible for the pathogenesis of ALCL.
Pathology - Research and Practice 02/2004; 200(10):669-79. · 1.21 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: This study analyzes the prevalence of T-cell non-Hodgkin's lymphoma (T-NHL) in a major referral center of Pakistan and its association with Epstein-Barr virus (EBV). Ninety-two cases of T-NHL were characterized on the basis of morphology, immunohistochemistry and genetic features. The prevalence of T-NHL was 22.2% of the total NHLs diagnosed during the eight years period (1992-1999). Polymerase chain reaction (PCR) technique was used to assess T-cell clonality in paraffin-embedded tissues of known T-NHL. Amplifiable DNA was isolated from all the cases, which were further studied for T-cell receptor (TcR)-beta, gamma, delta, and IgH chain gene rearrangements. Out of 92 cases 51 cases showed clonal product and 33 demonstrated polyclonal smear for beta, gamma, or delta chain genes, respectively, whereas 8 cases exhibited IgH chain gene rearrangement for FR2 region. This study demonstrated frequent presence of EBV in T-NHL (55.4%) by PCR, which were further tested for the localization of the virus by in situ hybridization (ISH). The extent of polymorphism in EBV genome was studied by single stranded conformation polymorphism (SSCP) technique for Bam HI E, K, N and Z regions. Hypervariability in Bam HI K, and N regions was noticeably higher compared to E or Z regions. In conclusion, our study demonstrated that the prevalence of T-NHL in Pakistan is slightly higher to that reported for Western communities. In addition, the frequency of EBV genome in T-NHL is intermediate as compared to other studies. No association was established between EBV variants differentiated on the basis of sequence heterogeneity in Bam HI K, N, E and Z regions with the manifestation of different subsets of T-NHL.
Leukemia and Lymphoma 06/2003; 44(5):807-13. · 2.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To analyze the spectrum of Non-Hodgkin's Lymphomas (NHL) presenting as skin lesions and to see the histological type, age, sex incidence and site predilection of different types of NHLs and to correlate our findings with western published data.
A retrospective descriptive case series study.
All NHLs (nodal and extra nodal) diagnosed between January 1991-December 2001 in the section of Histopathology, Department of Pathology, The Aga Khan University, Karachi were retrieved by using SNOMED coding system. Cases were reviewed and those presenting with skin involvement were selected. Further characterization of these tumours was done by morphology and Immunohistochemistry. Panel of antibodies included leukocyte common antigen (CD45), Pan B markers (CD20 and CD 79a), Pan T markers(UCHL1), Ki-1(CD30) and epithelial membrane antigen (EMA).
The total number of NHLs (nodal and extra nodal) diagnosed in our department during the study period of 10 years were 1610. Out of these, 133 cases (8.26%) of NHLs presented with skin involvement. Out of these 44.4% (n=59) were NHLs of B cell phenotype while 23.3% (n=31) cases were of T cell phenotype. Out of 133 cases, 14(10.5%) were of mycosis fungoides (MF), and 3 (2%) each were of anaplastic large cell lymphoma (ALCL:Ki-1), lymphoma and lymphoblastic lymphoma/leukemia. In 17.3% (n=23) cases the phenotype could not be ascertained. There were 97(73%) males and 36(27%) females with a M:F ratio of 2.7:1. All types of NHLs showed a site predilection for the head and neck region except MF which presented mainly as generalized body lesions.
NHLs presenting as skin lesions are more commonly of B cell phenotype followed by T cell phenotype (NOS) and mycosis fungoides. Probable reasons for the high frequency of cutaneous involvement by B-cell NHL are the advanced nature of the disease at the time of presentation and biological behavior of the tumors in our population.
Journal of the College of Physicians and Surgeons--Pakistan: JCPSP 02/2003; 13(1):29-32. · 0.34 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mycosis fungoides (MF) is an indolent T cell lymphoma that is distinguished from other lymphomas by its initial appearance on the skin. The histologic diagnosis of MF may be difficult because there is significant overlap in the histologic features of neoplastic T-cell infiltrates and inflammatory dermatoses. This T-cell neoplasm commonly occurs in a mixed, reactive background and can show only a subtle degree of cytologic atypia, rendering histologic diagnosis difficult. In this study MF constituted 0.86% of all non-Hodgkin s lymphoma (NHL) both T and B, as compared to the Western studies which have reported 0.5% prevalence for MF of all NHL. Polymerase chain reaction (PCR) technique was used to assess T-cell clonality in paraffin-embedded skin biopsies clinically and pathologically suspicious for early MF. Out of the 14 cases diagnosed as MF, amplifiable DNA was isolated from 6 cases, which were further studied for T-cell receptor (TcR) beta, gamma, and delta chain gene rearrangements. Clonal product was seen in 4 out of 6 cases for beta, gamma, and delta TcR chain genes. Association for Epstein Barr virus (EBV) was observed in 3 out of 6 cases (50%) of MF. Although these 3 cases were positive for EBV by PCR, but were negative by in-situ hybridization (ISH). No heterogeneity was noted in these 3 cases of MF for BamHI E, K, N, and Z regions of EBV. All six cases were negative for HTLV-1 (tax region) by PCR. It was concluded that the prevalence of MF in Pakistani population is comparable to the Western data, and that EBV association to MF cases was higher than in Western studies.
Pathology & Oncology Research 02/2002; 8(3):194-9. · 1.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mycosis fungoides (MF) is an indolent T cell lymphoma that is distinguished from other lymphomas by its initial appearance
on the skin. The histologic diagnosis of MF may be difficult because there is significant overlap in the histologic features
of neoplastic T-cell infiltrates and inflammatory dermatoses. This T-cell neoplasm commonly occurs in a mixed, reactive background
and can show only a subtle degree of cytologic atypia, rendering histologic diagnosis difficult. In this study MF constituted
0.86% of all non-Hodgkin’s lymphoma (NHL) both T and B, as compared to the Western studies which have reported 0.5% prevalence
for MF of all NHL. Polymerase chain reaction (PCR) technique was used to assess T-cell clonality in paraffinembedded skin
biopsies clinically and pathologically suspicious for early MF. Out of the 14 cases diagnosed as MF, amplifiable DNA was isolated
from 6 cases, which were further studied for T-cell receptor (TcR)-β, γ, and δ chain gene rearrangements. Clonal product was
seen in 4 out of 6 cases for β, γ, and δ TcR chain genes. Association for Epstein Barr virus (EBV) was observed in 3 out of
6 cases (50%) of MF. Although these 3 cases were positive for EBV by PCR, but were negative by in-situ hybridization (ISH).
No heterogeneity was noted in these 3 cases of MF for BainHI E, K, N, and Z regions of EBV. All six cases were negative for
HTLV-1 (tax region) by PCR. It was concluded that the prevalence of MF in Pakistani population is comparable to the Western
data, and that EBV association to MF cases was higher than in Western studies.
Pathology & Oncology Research - PATHOL ONCOL RES. 01/2002; 8(3):194-199.
